Proteins play crucial roles in virtually every biological process. The analysis and identification of proteins at the molecular level is, therefore, very significant and necessary for understanding the versatile biological activities of cells and, further, for developing new therapies for diseases.
Most proteins undergo some form of modification following translation. Most representative of the post-translational modification is phosphorylation. Most commonly occurring on tyrosine, serine, threonine, histidine and lysine residues, phosphorylation plays critical roles in the regulation of many cellular processes.
Particularly, reversible phosphorylation is known to be closely related to intra- and inter-cellular signaling events. Many of the proteins involved in cell signaling pathways are phosphorylated by kinases and dephosphorylated by phosphotases. Many reported that abnormal phosphorylation/dephosphorylation of specific proteins is causative in many diseases.
Fast and accurate screening for the irregularity of proteins in phosphorylation state is very helpful for understanding intra- or inter-cellular signaling events as well as contributing to the development of effectively diagnosing methods for diseases.
Tandem mass (MS/MS) spectrometry, which is one of the most potent protein analysis methods in current use, requires the hydrolysis of proteins to smaller mass peptides for analysis. The tandem mass spectrometry suffers some disadvantages when analyzing phosphopeptides, unlike the case of non-phosphopeptides. First, protein phosphorylation is usually reversible so that the sites on which phosphorylation can occur may be partially phosphorylated. Thus, in many cases, the amount of phosphopeptides obtained after the hydrolysis of proteins is present substoichiometrically. Second, the phosphoric acid groups present in phosphopeptides give a significant ionization suppression effect in cation mass spectrometry, which is generally adopted in peptide analysis. As a result, the detection sensitivity of phosphopeptides is very poor in relation to that of non-phosphopeptides. Finally, as phosphoric acid groups are chemically unstable, phosphopeptides may be partially dephosphorylated during their mass analysis. If dephosphorylation occurs, not only reduce the detection sensibility but also complicate the spectrum interpretation, resulting in an incomplete MS/MS sequencing analysis of peptide.
To avoid these problems, β-elimination of phosphoric acid groups is used. When being subjected to β-elimination in a basic solvent, phosphoserine and phosphothreonine residues of peptides or proteins are converted into dehydroalanine and β-methyldehydroalanine residues, respectively. In advance of mass spectrometry, phosphoric acid groups are removed from sample peptides or proteins. The elimination of phosphoric acid groups from peptides or proteins is accompanied by the removal of the above-mentioned problems, such as the ionization suppression due to the presence of phosphoric acid groups, and the partial dephosphorylation attributed to the chemical instability of phosphoric acid groups. However, the residues resulting from the dephosphorylation, that is, dehydroalanine and β-methyldehydroalanine are unstable, as well. In order to make the dephosphorylated peptide stable, the residues are rendered to undergo Michael addition reactions with properly-designed, various nucleophiles. Consequently, the problems of phosphopeptides on mass spectrometry can be solved through dephosphorylation, and the analysis of phosphopeptides is facilitated by introducing tag with versatile information such as specific mass number into the dephosphorylated amino acid residues.
Tags, used in β-elmination/Michael addition reaction, generally contain sulfhydryl functionality. The tags can also have neutral functional groups or basic functional groups such as amine groups promoting the generation of cations in peptides. Thus the mass sensitivity of the tagged peptides is exceptionally improved. The tagged peptide derivatives are also so stable under general mass spectrometric conditions that accurate amino acid sequencing can be achieved using MS/MS spectrometry.
Another advantage of phosphospecific tagging method is that relative quantification between samples obtained from different sources can be achieved by using stable isotope-coded tag. When compared to intact tag, stable isotope-coded tag shows the same reactivity in the β-elimination/addition reaction and no different analysis sensitivity in mass spectrometry. Only difference between intact tag and stable isotope-coded tag in mass spectrometry is mass values read on mass spectra of each peptide derivatives. Thus the intact tag and the stable isotope-coded tag are used together but separately for tagging of each sample so that relative quantification can be achieved between samples obtained from different sources.
A further advantage provided by the β-elimination/addition reaction is that only tag derivatives of phosphopeptides can be selectively purified and concentrated by introducing an appropriate functional group having affinitive interactivity to tag. For example, a tag having biotin structure is used the purpose (Oda, Y. et al., 2001, Nature Biotechnology, 19, 379-382). When a biotin group is linked through ethanedithiol to a dephosphorylated residue, streptavidin which can affinitively interact with biotin can be used to selectively isolate and concentrate the tagged peptides formerly phosphopeptides from sample. Various neucleophiles containing a thiol group are used to label phosphopeptides and proteins.
None the less, there is a need of an improved mass spectrometry method for effectively analyzing phosphoproteins which are present in the body in trace amounts or in substoichiometric amounts due to partial phosphorylation as well as the phosphorylated sites of the proteins.